The Hering-Breuer reflex and hypoxic chemoreflex are two basic tenets of respiratory physiology and medicine that have been widely studied at the systems level. However, the central mechanisms mediating these important reflexes remain largely unknown - as the underlying complex processes do not lend themselves readily to systematic analyses using traditional coarse-grained neuronphysiological and modeling approaches. A major hurdle limiting progress is the lack of detailed information about the cytoarchitecture of brainstem neurons involved in respiratory afferents processing. To circumvent this difficulty, the proposed New Investigator project will employ a novel systems biology research design that builds upon two synergistic fine-grained approaches. First, recent modeling studies from our group reveal that the Hering-Breuer reflex and hypoxic chemoreflex may share surprisingly similar pontomedullary pathways with similar afferents processing mechanisms albeit with opposite respiratory effects. These working models lay out a valuable blueprint that is critical for systematic experimental mapping of these neuronal pathways and for hypotheses testing. Second, to precisely delineate the cytoarchitecture of these afferents processing pathways we will employ two advanced techniques that allow us to characterize the afferent-efferent neurotransmission processes in individual functionally-identified pontine respiratory neurons: (1) microiontophoresis of selected neurochemicals in order to define the transmitters- receptors involved in neuronal afferent (dendritic) neurotransmission; and (2) juxtacellular labeling in combination with immunohistochemistry or in situ hybridization in order to define the specific neurotransmitters involved in neuronal efferent (axonal) transmission. These studies will be combined with antidromic activation techniques to trace the functional interconnections between different components of the afferents processing pathways. In harness with our working models, these fine-grained and coarse-grained assays make it possible to precisely, systematically and cost-effectively map the neuronal structure-function relationships of the model-predicted afferents processing pathways at the single-cell level. In this project, we will target specific respiratory neurons in rat pneumotaxic nuclei in the dorsolateral pons that have recently been implicated in co-mediating the Hering- Breuer reflex and hypoxic chemoreflex. Our specific aims are to characterize the afferent (Aim 1) and efferent (Aim 2) neurotransmission phenotypes of pneumotaxic neurons in these pontine nuclei that contribute to the Hering-Breuer reflex and hypoxic chemoreflex and other respiratory-related events. Together with our previous electrophysiological and neurotracing studies, these critical cell-specific structure-function data will allow us to critically test extant neural network models of pontine modulation of breathing in the literature and to enable future refinement of these models. The proposed studies represent the first viable (techniques-driven) and systematic (models-driven) approach to defining the cytoarchitecture of pneumotaxic processes mediating the Hering-Breuer reflex and hypoxic chemoreflex, for which little information is currently available. PUBLIC HEALTH RELEVANCE: Breathing is a vital function that is controlled by the brainstem according to the needs of the body. This project studies how various neurochemicals regulate the control of breathing in the brainstem. Understanding this question will help us to develop better pharmacological treatments for respiratory diseases such as respiratory failure, sudden infant death, sleep apnea, and various other respiratory disorders and to better cope with mountain sickness at high altitude.